Method for expansion of double negative regulatory T cells

ABSTRACT

There is provided herein a method for expanding human CD4-CD8- regulatory T cells (DN Tregs) from a population of cells comprising DN Tregs, comprising: culturing the population of cells with artificial antigen presenting cells (APCs), preferably the DN Tregs are αβ-TCR-CD56- or alternatively γ8-TCR+.

RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. 371 ofInternational Application No. PCT/CA2016/000136 filed May 11, 2016,which claims priority to U.S. Provisional Application Nos. 62/159,561and 62/237,050 filed on May 11, 2015 and Oct. 5, 20151 respectively, allof which are incorporated herein by reference in their entirety.

FIELD

The present invention relates to regulatory T cells, and in particularmethods of expanding the double negative subset of regulatory T cells.

BACKGROUND

Compelling evidence has shown the importance of regulatory T cells(Tregs) in controlling various diseases including autoimmune diseases,allograft rejection, graft vs. host disease (GVHD), infections andcancer^(1-7,9). Tregs consist of many distinct subsets, althoughnaturally occurring CD4+CD25+Foxp3⁺ Tregs (nTregs) are the mostextensively studied⁸′⁹. Applicant refers also to its issued U.S. Pat.No. 9,018,004 for expanding double negative T cells.

Applicant previously identified and characterized another subset ofTregs which express αβ-TCR but not CD4, CD8, or NK cell marker NK1.1 (inmice) or CD56 (in humans), and termed double negative regulatory Tcells, or DN Tregs in short¹. DN Tregs comprise 1% of mature CD3+ Tcells in rodents¹¹ and humans¹⁰. It has been demonstrated that adoptivetransfer of DN Tregs can establish and maintain donor-specific toleranceto allogeneic islet, skin and heart, as well as xenogeneic heartgrafts¹¹⁻¹⁷. DN Tregs can also attenuate allogeneic CD4+ and CD8+ Tcell-mediated GVHD¹⁸⁻²¹, and control autoimmune diabetes,lymphoproliferative syndrome and infection^(12,22-24.) Human DN Tregshave similar cell surface marker expression and produce similarcytokines as their murine counterparts, and can also suppressAg-specific immune responses in vitro^(10,25). In addition, a higherfrequency of DN Tregs in patient blood after hematopoietic stem celltransplantation correlates with mild GVHD^(26,27). Collectively, thesefindings demonstrate that DN Tregs can control the development andpathogenesis of various diseases by suppressing antigen-specific immuneresponses.

Given that numerous pre-clinical studies have demonstrated theimportance of Tregs in controlling various diseases, many attempts havebeen made to produce large numbers of human Tregs for clinical use. Itis now possible to produce therapeutic numbers of anti-CD3-activatedpolyclonal nTregs for clinical trials²⁸. It has been shown that human DNTregs can be generated by stimulation with allogeneic monocyte-deriveddendritic cells¹⁰. However, the number of human DN Tregs generated inthis way is very limited.

SUMMARY

In an aspect, there is provided a method for expanding human CD4⁻ CD8⁻regulatory T cells (DN Tregs) from a population of cells comprising DNTregs, comprising: culturing the population of cells with antigenpresenting cells (APCs), preferably the DN Tregs are CD56⁻, αβ-TCR+,γδ-TCR+, or combinations thereof.

In an aspect, there is provided a population of DN Tregs obtained by themethods described herein.

In an aspect, there is provided a use of DN Tregs for the treatment ofcancer, preferably leukemia or lung cancer.

In an aspect, there is provided a use of DN Tregs in the preparation ofa medicament for the treatment of cancer, preferably leukemia or lungcancer.

In an aspect, there is provided a method of treating cancer in asubject, preferably leukemia or lung cancer, comprising administering tothe subject a therapeutically effective amount of DN Tregs.

In an aspect, there is provided a use of DN Tregs for the treatment orprevention of allograft rejection, GVHD, or an autoimmune disease.

In an aspect, there is provided a use of DN Tregs in the preparation ofa medicament for the treatment or prevention of allograft rejection,GVHD, or autoimmune diseases.

In an aspect, there is provided a method of treating or preventingallograft rejection, GVHD, or an autoimmune disease, comprisingadministering to the subject a therapeutically effective amount of DNTregs.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described, by way ofexample only, with reference to the attached Figures.

FIG. 1 shows isolation and expansion of DN Tregs. (A) DN Tregs wereenriched by staining PBMCs with CD4-FITC, CD8-FITC, CD56-FITC,TCRγδ-FITC Abs and depleting the stained cells using anti-FITC magneticbeads and MACS technology. Representative flow cytometry histograms ofpre- and post-sort are shown. (B) Fold increase in DN Tregs cells after21 days of ex vivo expansion (n=8). (C) Purity of DN Tregs was assessedby flow cytometry on day 0 and day 21. Representative figure is shown.

FIG. 2 shows that DN Tregs suppress proliferation of autologous CD4+,CD8+ and CD19+ cells. Purified naïve CD4+, CD8+ or CD19+ cells werelabeled with CFSE and co-cultured with αCD3/CD28 beads or F(ab′)2fragment of goat anti-human IgM, respectively, in the presence of exvivo-expanded DN Tregs for 4 days. Proliferation was measured by CFSEdilution. The data are expressed as mean percentage inhibition of threereplicates. Error bars represent SD. The experiment was done intriplicates, and repeated 8 times (A,B) or 5 times (C) with cellsobtained from different donors. Similar results were observed.

FIG. 3 shows that DN Tregs suppress secretion of IFN-γ by CD4+ and CD8+T cells and IL-10 by CD4+ T cells. The concentration of IFN-γ (A) andIL-10 (B) was measured in the supernatants obtained from a 4-daysuppression assay of DN Tregs and CD4+ or CD8+ T cells co-cultures, at4:1, supressor-to-responder ratio. The values represent mean±SD of 3replicates. Similar results were obtained from 3 different suppressionassays, each executed with a different donor. * p<0.05, ** p<0.01, ***p<0.001, n.d. non-detected.

FIG. 4 shows that Rapamycin-treated DN Tregs manifest augmentedregulatory function. Ex vivo-expanded DN Tregs were pre-incubated withRapamycin for 2 h, washed extensively and used as suppressor cells in asuppression assay against autologous CD4+ and CD8+ T cells stimulatedwith αCD3/CD28 beads. On day 3, proliferation of responder cells wasquantified by CFSE dilution. The histograms represent proliferation ofresponder cells at 1:1, supressor-to-responder cells ratio. The numberrepresent percentage of proliferating responder cells. Similar resultswere observed in 3 independent experiments.

FIG. 5 shows that DN Tregs delay onset of xenogeneic GVHD. 6 week oldNSG mice were sublethally irradiated (250cGy) and i.v. injected withPBMCs (n=23) obtained from a healthy human donor or DN Tregs (n=14) exvivo-expanded from the same donor. Treated mice were infused with PBMCsfollowed by three injections of DN Tregs on day 0, 3 and 7 (n=14). Miceinjected with PBS alone (n=12) were used as controls. Mice weremonitored daily for the signs of GVHD. (B) Survival curves of recipientmice. Statistical differences between the curves were compared usinglog-rank test PBMC vs Treated 3 inj., p<0.001; PBMC vs DN Treg, p<0.001;PBMC vs PBS, p<0.001. (C) Weight of mice was monitored daily to assessseverity of GVHD. Mice that lost >20% of their initial body weight wereeuthanized Kruskal-Wallis test was used to determine statisticalsignificance. Survival and weight monitoring data were pooled from threeseparate experiments.

FIG. 6 shows that ex vivo-expanded DN Tregs can kill human cancer cells.Cytotoxicity against human leukemic (A) or lung cancer (B) cell lines byex vivo-expanded human DN Tregs was determined by flow cytometry killingassay. In short, cancer cells were labeled with PKH and co-cultured for2 h (A) or 16 h (B) with DN Tregs at the indicated ratios. Specifickilling of cancer cells was determined by the proportion of cellsremaining alive after co-incubation with DN Tregs. Similar results wereobtained from 3 different donors.

FIG. 7 shows tracking and in vivo proliferation of expanded DN Tregs inNSG mice. Ex vivo-expanded DN Tregs were stained with CFSE and injectedinto sublethally irradiated (250 cGy) NSG mice. On days 1, 3, 5, 7 and10, 2 to 3 mice per group were sacrificed and the following tissues wereharvested: blood, bone marrow, spleen, lymph nodes, kidneys, liver andlungs. (A) The frequency of DN Tregs from each tissue was assessed byflow cytometry. (B) Analysis of proliferation of DN Tregs was assessedby CFSE dilution. Here is an example of DN Tregs isolated from thespleen.

FIG. 8 shows a) Purified naïve CD8+(right) cells (1×10⁵/well) werelabeled with CFSE and co-cultured with anti-CD3/CD28 beads in theabsence or presence of ex vivo expanded allogeneic DNT (8×10⁵/well,bottom panels) for 3 days. Proliferation of CD8+ T cells was measured byCFSE dilution. Representative histograms are shown. Numbers representthe frequency of proliferating cells. Filled histograms are the CD4+ orCD8+ T cells cultured in media only. The experiment was performed intriplicates. b) Sublethally irradiated NSG mice were intravenouslyinjected with 1×10⁷ human PBMCs, DNTs or PBS (n=5 per group). On days 2,6, 10, and 14 after injection, mouse body weight was measured. The graphshown is representative of results from five DNT and five PBMC donors.d) NSG mice injected with 5×10⁶ human PBMC were infused with PBS (•) or2×10⁷ allogeneic DNTs on days 0, 3, and 6 after PBMC injection.

FIG. 9 shows that DNT cells do not kill or interfere with normalhematopoetic cells. a) Freshly isolated healthy BM cells were culturedeither alone or with ex vivo expanded autologous DNT cells at a 4:1ratio. After 12 and 24 hours of culture, cells were stained withanti-human CD34, CD3, CD33 antibodies and viability dyes, AnnexinV andeF450. The viability of CD34+CD3- (left) and CD33+CD3- (right) cellswere determined using flow cytometry. (b-c) Cytotoxicity of allogeneicDNTs expanded from three HDs against CD34-AML, OCl/AML3, primary AMLpatient blasts, 110164 and 090596, and normal PBMCs from two HDs (b) oragainst CD34+AMLs, 130723, 090240, and 130624 and HSPCs from twodifferent HDs (c) at 4—to1 effector-to-target ratios was determinedusing the 2-hour flow-based killing assay. d) Freshly harvested BM cellswere injected directly into the left femurs of NSG mice (5×106/mouse,n=8). 1 day later, half of the mice were injected i.v. with ex vivoexpanded autologous DNTs (5×106/mouse), and half (n=4) were injectedwith PBS as controls. BM from each mouse was harvested 5 days later,stained with anti-human CD34 antibody and the cell viability dyesAnnexin V and eF450, and analyzed by flow cytometry. Bar graph shows thetotal number of human CD34+ cells in mouse BM in both groups. Error barsrepresent mean±SD. (e and f) CD133+CD34+human HSPCs were intravenouslyinjected into sublethally irradiated NSG mice (3×105 cells/mouse, n=13).Eight weeks post HSPC injection, 7 of the 13 mice were intravenouslyinjected with 107 ex vivo expanded allogeneic DNTs and the rest wereinjected with PBS. To determine chimerism originating from the HSPCpopulation, cells from BM, spleen, and PB were harvested 8 weeks afterDNT injection and were stained with anti-mouse CD45, anti-human CD45,CD3, CD19, CD11b, CD56, CD33, and CD34 antibodies. The percentage ofhuman leukocytes e) and its subsets f) were determined by flow cytometryanalysis. Horizontal bars represent the mean value and the error barsrepresent SEM of each group.

DETAILED DESCRIPTION

In order to accelerate the clinical use of human DN Tregs, the presentmethod was developed that allows for a large-scale (˜4000 fold) ex vivoexpansion of human DN Tregs and it was demonstrated that exvivo-expanded human DN Tregs can suppress CD4+ and CD8+ T cell, andCD19+B cell proliferation and kill various cancer cell lines in vitro.Human DN Tregs does not induce xenogeneic GVHD after infusion intoimmunodeficient mice and can delay an onset of xenogeneic GVHD inducedby human PBMC. There is therefore provided herein a method for largescale ex vivo expansion of dual functional human a13-TCR+CD4⁻ CD8⁻ CD56⁻regulatory T cells.

In an aspect, there is provided a method for expanding human CD4⁻ CD8⁻regulatory T cells (DN Tregs) from a population of cells comprising DNTregs, comprising: culturing the population of cells with antigenpresenting cells (APCs), preferably the DN Tregs are αβ-TCR+CD56⁻,γδ-TCR+, or both.

The term “double negative Treg cell” may mean a T lymphocyte thatexpresses CD3 but lacks the cell surface expression of CD4 and CD8. TheDN T cells will express a T-cell receptor (TCR) which can be either theαβ or the γδ TCR.

An antigen presenting cell (APC) or accessory cell is a cell thatdisplays typically foreign antigens complexed with majorhistocompatibility complexes (MHCs) on their surfaces; this process isknown as antigen presentation. T-cells may recognize these complexesusing their T-cell receptors (TCRs). These cells process antigens andpresent them to T-cells. Artificial antigen presenting cells aresynthetic versions of APCs and are made by attaching the specific T-cellstimulating signals to various macro and micro biocompatible surfaces.Cell-based artificial antigen presenting cells are also known in theart. Cell based aAPCs can be made, for example, by transfecting cells toexpress specific antigens, and optionally co-stimulatory or cellsadhesion molecules. A person skilled in the art would understand thescope and types of APCs (and associated co-stimulatory or cells adhesionmolecules) that could be used in the present methods, based on thepresent disclosure.

In an embodiment, the APCs are artificial APCs displaying anti-CD3antibodies and at least one cell surface adhesion molecule forimmunological synapse. Optionally, the cell surface adhesion molecule isat least one of CD54 and CD58.

In some embodiments, the APCs further express a co-stimulatory molecule,preferably at least one of CD80, CD86, CD83, 4-1BBL, OX40L, ICOSL,CD40L, and CD28 antibody.

In some embodiments, the APCs do not express inhibitory molecules, suchas PDL1, PDL2, B7H3 and B7H4.

In some embodiments, the APCs further express at least one of M-CSF,IL-6, IL-8, TGF-β and MIP-1a.

In some embodiments, the APCs further express IL-7, IL-15 and/or IL-2.

In some embodiments, the APCs comprise K562-based artificial APCs,preferably as set forth in Tables 1 and 2.

TABLE 1 K562-based artificial APCs for natural killer cell and Tregclinical studies K562- based Target cell aAPC and for ex- transducedpansion molecules Target disease and notes Phase Status Ref. K562-Relapsed high Autologous I Open NCT01212897; mbIL15- risk multiple NKcell plus University of 41BBL: myeloma bortezomib Arkansas 4-1BBL,Asymptomatic Autologous I Open NCT01884688; membrane- multiple NK cellsUniversity of bound myeloma post Arkansas IL-15 standard therapy aAPCB-cell chronic Allogeneic I Open NCT01619761; (clone #9): lymphocytic NKcells DACC CD64, leukemia derived from CD86, undergoing cord blood4-1BBL, umbilical cord truncated SCT CD19, Multiple Allogeneic I OpenNCT01729091; membrane myeloma NK cells MDACC bound undergoing derivedfrom IL-21 umbilical cord cord blood SCT Myeloid Allogeneic I OpenNCT01823198; leukemia NK cells MDACC undergoing derived from SCT cordblood KT64/86: Advanced Natural I Open NCT00602693; CD64, hematologicTreg from University of CD86 malignancies umbilical Minnesota withumbilical cord cells cord SCT expanded with aAPC loaded with OKT3 aAPC,artificial antigen presenting cell; CAR, chimeric antigen receptor;DFCI, Dana-Farber Cancer Institute; GFP, green fluorescence protein;EBV, Epstein-Barr virus; mIL-15, membrane bound IL-15; mIL-21, membranebound IL-21; MDACC, M.D. Anderson Cancer Center; MTD, maximum tolerateddose; NCI, National Cancer Institute; PMCC, Princess Margaret CancerCentre; SCT, stem cell transplant

TABLE 2 K562-based artificial APCs or anti-tumor T-cell clinical studiesK562-based aAPC and Target cell for transduced expansion andClinicalTrials.gov molecules Target disease notes Phase StatusIdentifier aAPC-A2, Advanced Autologous aAPC- I Closed NCT00512889;clone 33: HLA melanoma generated MARTI T DFCI; future class I, CD80,cells studies at Princess CD83 Margaret 7F11ECCE: Advanced TIL fortransfer II Closed NCT01369875; CD64, 4-1BBL melanoma after NCIlymphodepletion using aAPC loaded with OKT3 K562cs: CD32, EBV-positiveEBV-specific T cell I Open NCT01555892; CD80, CD83, lymphoma lines, aAPCused for Texas Children's CD86, 4-1BBL costimulation, not and Methodistantigen-specific Hospital stimulation aAPC (clone B-Lineage AutologousCD19- I Open NCT00968760; #4): CD64, Lymphoid specific CAR T cells MDACCCD86, 4-1BBL, Malignancies Allogeneic CD19- I Open NCT01497184;truncated After Auto-SCT specific CAR T cells MDACC CD19, B-Lineagederived from cord membrane- Lymphoid blood bound IL-15 MalignanciesAllogeneic CD19- I Open NCT01362452; after Umbilical specific CAR Tcells MDACC Cord SCT Autologous CD19- I Open NCT01653717; B-Lineagespecific CAR T cells MDACC Lymphoid Malignancies after allo-SCT B-cellChronic Lymphocytic Leukemia

In alternate embodiments, the APCs are autologous APCs, preferably,dendritic cells, monocytes or activated B cells.

In some embodiments, the culturing is additionally in the presence ofIL-2.

In some embodiments, the culturing is additionally in the presence ofIL-7 and/or IL-15.

In some embodiments, the method further comprises activating thepopulation with anti-CD3 antibodies prior to culturing with the APCs.Optionally, the activating is with anti-CD3 antibodies cross-linked, orotherwise attached, to a surface or alternatively, with soluble anti-CD3antibodies.

In some embodiments, the activating is sequentially with anti-CD3antibodies cross-linked, or otherwise attached, to a surface and solubleanti-CD3 antibodies.

In some embodiments, the method further comprises at least partiallydepleting the population of non-DN Tregs cells prior to culturing and/oractivating. Optionally, the population is depleted based on a cellsurface marker, preferably using antibodies to the cell surface markerbound to magnetic beads. The cell surface marker is preferably at leastone of CD4+, CD8+, CD56+ and γδ-TCR+. Following culturing the populationmay be further depleted this manner.

In some embodiments, the population comprises peripheral bloodmononuclear cells (PBMCs) or cord blood mononuclear cells (CBMCs).

In some embodiments, the method further comprises incubating theexpanded DN Tregs with at least one inhibitor of the PI3K/AKT/mTORpathway.

Four classes of inhibitors that will inhibit the PI3K/AKT/mTOR pathwayare currently identified. Many such inhibitors are known in the art andcurrently in clinical trials. In some embodiments, those upstream of thesignaling cascade are used with the method described herein. A review byDienstmann et al.³⁰ includes inhibitors that are currently in clinicaltrials for cancer treatment.

In some embodiments, the inhibitor is a mTOR inhibitor, dual PI3K/mTORinhibitor, AKT inhibitor, or Pan-class I and isoform-specific PI3Kinhibitors. Preferably, the inhibitor is a Rapalog, further preferably,rapamycin, deforolimus, emsirolimus, everolimus, ridaforolimus,temsirolimus or a mTORC1/mTORC2 dual inhibitor.

In other embodiments, the inhibitor is Wortmannin, LY294002, PKI-179, orAkt inhibitor IV.

In an aspect, there is provided a population of DN Tregs obtained by themethods described herein.

In an aspect, there is provided a use of DN Tregs for the treatment ofcancer, preferably leukemia or lung cancer.

In an aspect, there is provided a use of DN Tregs in the preparation ofa medicament for the treatment of cancer, preferably leukemia or lungcancer.

In an aspect, there is provided a method of treating cancer in asubject, preferably leukemia or lung cancer, comprising administering tothe subject a therapeutically effective amount of DN Tregs.

In an aspect, there is provided a use of DN Tregs for the treatment orprevention of allograft rejection, GVHD, or an autoimmune disease. Giventhe examples provided herein, it is anticipated that DN Tregs could beused to treat or prevent allograft rejection, GVHD, or an autoimmunedisease, preferably graft rejection, in humans. In some embodiments, theDN Tregs are αβ-TCR+. In other embodiments, the DN Tregs are γδ-TCR+.

In an aspect, there is provided a use of DN Tregs in the preparation ofa medicament for the treatment or prevention of allograft rejection,GVHD, or autoimmune diseases.

In an aspect, there is provided a method of treating or preventingallograft rejection, GVHD, or an autoimmune disease, comprisingadministering to the subject a therapeutically effective amount of DNTregs.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desired result,e.g. to treat a tumor.

The term “treating” includes, but is not limited to, alleviation oramelioration of one or more symptoms or conditions of a disease orcondition (such as cancer, autoimmune disease, allergy, infection etc.),diminishment of extent of disease, stabilized state of disease,preventing spread of disease, delaying or slowing of diseaseprogression, and amelioration or palliation of the disease state,remission whether detectable or undetectable and/or prolonged survivalas compared to expected survival if not receiving treatment.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the cells is combined in a mixture with a pharmaceutically acceptablevehicle. Suitable vehicles are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th ed.,Mack Publishing Company, Easton, Pa., USA 2000). On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

An effective amount of the composition may vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe ability of the cells to elicit a desired response in the individual.Dosage regime may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

The pharmaceutical compositions of the invention may include otheractive agents that are useful in treating the disease or condition to betreated. For example, in the treatment of a tumor, other anti-canceragents may be administered either in the same composition or in aseparate composition.

The advantages of the present invention are further illustrated by thefollowing examples. The examples and their particular details set forthherein are presented for illustration only and should not be construedas a limitation on the claims of the present invention.

EXAMPLES

Methods

Ex Vivo Expansion of DN Tregs

DN Tregs comprise about 1% of peripheral T cells of healthy adults. Toexpand human DN Tregs, PBMCs were isolated by Ficoll density gradientand DN Treg population was enriched by depleting population of T cellsand NK cells that would be prone to outgrow using this expansion method.In short, PBMCs were stained with FITC conjugated antibodies to CD4,CD8, CD56 and TCRγ6 markers, and anti-FITC magnetic beads (MiltenyiBiotec) (FIG. 1A). From 50-80 ml of blood, we were able to obtain5-25×10⁵ DN Tregs. After sorting, cells were resuspended in RPMI mediasupplemented with 10% FBS and recombinant human (rh)IL-2. In order toactivate DN Tregs, cells were seeded in the tissue culture plate coatedwith CD3 monoclonal antibody (OKT3). On day 3 of culture, cells wereharvested, washed and cultured for another 4-7 days in the presence ofrhlL-2 and rhlL-7 and/or rhIL15 followed by co-culture with lethallyirradiated (150 Gy) aAPCs in the presence of rhlL-2 and rhlL-7 and/orrhIL15. DN Tregs were used for functional assays on day 22. Thephenotype of the cells was assessed regularly to monitor for potentialoutgrowth of CD4+, CD8+, CD56+ or TCRγ6 cells. If other cells besides DNTregs were present, they were depleted using magnetic beads sorting. Byday 22, the average purity of DN Tregs was 95.48±2.49% (FIG. 1C). Wewere able to obtain 1-18×10⁸ cells, with the average fold expansion of3900±2835 (FIG. 1B).

Results and Discussion

Ex Vivo Expanded DN Tregs are Potent Suppressors In Vitro

Previously, human DN Tregs that were activated by allogeneic DCs havebeen shown to suppress proliferation of CD4+ T cells in antigen- andallo-specific manner³¹⁻³⁴ However, whether poly-clonally activated andex vivo-expanded DN Tregs can retain their suppressive function has notbeen studied previously. Thus, we evaluated the ability of exvivo-expanded DN Tregs to inhibit proliferation of autologous CD4+ andCD8+ T cells, as well as CD19+B cells. To this end, CFSE-labeled T cellsor B cells were stimulated with αCD3/CD28 coated beads, or F(ab′)2fragment of IgM, respectively, and co-cultured in the presence orabsence of DN Tregs at increasing ratios. After 4-5 days of co-culture,proliferation of responder cells was assessed by CFSE-dilution usingflow cytometry. DN Tregs were found to potently suppress proliferationof CD4+ T cells (FIG. 2A), CD8+ T cells (FIG. 2B) and CD19+B cells (FIG.2C) in a dose-dependent manner. Interestingly, suppression of CD4+ Tcells by DN Tregs was consistently stronger than suppression of CD8+ Tcells for all the donors that we have tested. Furthermore, CD4+ T cellsproduce IL-10 (FIG. 3B) and both CD4+ and CD8+ T cells produced highamounts of IFN-γ upon stimulation with αCD3/CD28 beads (FIG. 3A).However, upon addition of DN Tregs to cell cultures at 4:1,suppressor-to-responder ratio, the amount of IFN-γ and IL-10 released bythe responder cells was significantly reduced, thus suggesting DNTreg-mediates suppression of activation of responder cells.

Rapamycin Augmented Immunosuppressive Function of DN Tregs

Rapamycin is an mTOR inhibitor and has been shown to facilitate nTregexpansion and regulatory function³⁵. Therefore, we investigated whetherthe inhibition of Akt/mTOR pathway by Rapamycin could improveimmunosuppressive function of DN Tregs. For this purpose, exvivo-expanded DN Tregs were pre-incubated with Rapamycin for 2 hours,extensively washed and used in the suppression assay. Blockade of theAkt/mTOR pathway by Rapamycin rendered DN Tregs more immunosuppressiveas they inhibited proliferation of autologous CD4+ and CD8+ T cells to agreater extent than non-treated DN Treg, as assessed by CFSE dilution(FIG. 4 ). Suppression of CD4+ T cells increased significantly by almost52±2% at the suppressor-to-responder ratio of 1:1. Since DN Tregs alwaysexerted more modest suppression against CD8+ cells, Rapamycin-treated DNTregs demonstrated significant improvement in their suppressive activityby 37±1% at 1:1, suppressor-to-responder ratio.

Ex vivo expanded DN Tregs delayed onset of xenogeneic GVHD in NSG miceFor evaluation of the in vivo effects of ex vivo expanded human DNTregs, PBMCs were freshly isolated from peripheral blood of the samedonor and infused into sub-lethally irradiated NSG mice to inducexenogeneic GVHD. To ameliorate GVHD, we proposed treatment regimen thatinvolved injection of 3 doses of DN Tregs over the span of first weeksince induction of GVHD. Mice receiving PBMC injection developed GVHDand all animals were dead by 31 days with the mean survival time (MST)of 19 days (FIG. 5A). In most cases, death was attributable to severweight loss (FIG. 5B). Recipients of adoptive transfer of 3 doses of DNTregs significantly delayed onset of GVHD (MST=29, p<0.001), with thelast subject surviving for 77 days. Importantly, mice injected solelywith DN Tregs remain healthy, as all mice remained alive 100 days postinjection without development of clinical signs of GVHD (FIG. 5B), thushighlighting the safety and lack of immunogenicity of DN Tregs.Furthermore, in vivo DN Tregs traffic to many hematopoietic and lymphoidtissues, including peripheral blood, bone marrow, spleen and lymphnodes, as well as other organs including lung, liver and kidneys (FIG.7A). DN Tregs proliferate in vivo and can be found in the tissues 10days post injection (FIG. 7B).

Ex Vivo Expanded DN Tregs can Kill Human Cancer Cells

Previously, we demonstrated that both mouse TCRαβ+ and TCRγδ+populations sorted from DN T cells expanded from peripheral blood of AMLpatients in complete remission were cytotoxic against autologous andallogeneic leukemic blasts in vitro⁶. Therefore, we wanted to testwhether DN Tregs expanded by the means of a novel protocol alsodemonstrate cytotoxic function against various human cancer cell lines.To assess DN Treg-induced killing of cancer, a flow cytometry-basedkilling assay was adapted in which target cells were stained with PKH-26before co-culture with DN Tregs. The percentage of cytotoxicity in thePKH-26-gated cell population was calculated as described in methods. DNTregs exerted dose-dependent cytotoxicity against all human primary lungcancer cell lines that were tested: 186-144, 426-177 and H125 lines(FIG. 6B). It was also found that DN Tregs effectively killed leukemicMV4-11 and K562 cells lines, but were less cytotoxic toward AML-3 andKG1a cells lines (FIG. 6A). These findings demonstrate that DN Tregs notonly exhibit immunoregulatory function, but also are cytotoxic tocancer.

The protocol described above allows for large-scale ex vivo expansion ofhuman DN Tregs. DN Tregs are co-cultured with irradiated artificial APC(human K562 cell line with surface expression of a transduced membranousform of anti-CD3 mAb, CD80, CD83, and 4-1BBL)²⁹, in the presence ofcombination of cytokines. Using this protocol, up to ˜10⁹ huDN Tregs(˜4000-fold expansion) from 50-80 ml blood were obtained in 3 weeks(FIG. 1B) with very high purity (FIG. 1C). Importantly, these exvivo-expanded DN Tregs can suppress in vitro the proliferation of bothCD4+ and CD8+ T cells (FIG. 2A-B), as well as CD19+B cells (FIG. 2C)stimulated in a polyclonal manner. Moreover, DN Tregs were found to killvarious leukemic and lung cancer cell lines. While infusion of humanperipheral blood mononuclear cells (PBMC) into immunodeficient NSG miceinduces severe acute xenogeneic GVHD, infusion of ex vivo expanded humanDN Tregs did not cause GVHD or tissue damage in recipients. Furthermore,treatment with DN Tregs significantly delayed an onset of xenogeneicGVHD in humanized mouse model. The ability to obtain large numbers offunctional DN Tregs not only makes it possible, for the first time, tostudy their function and mechanisms of action in vivo, but alsoindicates the possibility of using ex vivo-expanded human DN Tregs totreat various diseases such as allograft rejection, GVHD, autoimmunediseases and malignant diseases.

Graft-Vs-Leukemia (GVL) and Graft-Vs-Host Disease (GVHD)

Hematopoietic stem cell transplantation from a matched related orunrelated donor is considered standard of care in eligible patients withhigh risk clinical, cytogenetic and molecular features.(S1, S2) This isalso the only potentially curative treatment for AML patients withrelapsed disease.(S3-S7). The role of HSCT in patients with positive MRDstatus is also increasingly important.(S8) The efficacy of HSCT in AMLis based on the intensity of the conditioning regimen as well as theimmune mediated anti-tumour activity of the graft-vs-leukemia (GVL)effect.(S9) This effect was identified when it was noted that HSCTrecipients who developed acute and chronic GVHD had a lower incidence ofrelapse.(S10) This effect is mediated through the action of cytotoxicdonor T-cells and NK cells. However, donor T cells commonly recognizenormal host tissue and cause detrimental, and even lethal GVHD(S11,S12), compromising the overall benefit of allo-HSCT on patient survival.The toxicity of the conditioning regimens and complications of GVHDresult in significant treatment related mortality. Over time advances inantimicrobial therapy, reduction of regimen toxicity and improvements inthe prevention and treatment of GVHD have considerably reduced but noteliminated non-relapse mortality.(S13) Furthermore, broad application ofallo-HSCT is limited by recipient fitness and availability of suitabledonors in a timely manner. Reduced intensity conditioning regimens weredevised in an effort to capitalize on the GVL effect as a therapeuticmeasure in the absence of the toxicity associated with intensemyeloablative conditioning regimens.(S14, S15) Further refinement ofthis concept based around the harnessing of the GVL effect to achieveeffective disease control while reducing the risk of GVHD has resultedin the development of immune cell therapies.(S16-S18)

Mouse DNT Cells and their Role in GVL and GVHD

DNT cells are mature T lymphocytes that comprise ˜1% of peripheral bloodmononuclear cells (PBMC) in mice, rats and humans. DNT cells express CD3and αβ—or γδ-TCR, but not CD4, CD8 or NK cell markers nor bind invariantnatural killer T (iNKT) cell specific αGalCer-loaded CD1d tetramers,thus differ from conventional T cells, NK cells and NKT cells. The Zhanglab was the first to characterize DNT cells(S19) and showed in vivo thatunlike conventional CD4+ or CD8+ T cells, infusion of mouse allogeneicDNT cells not only did not induce GVHD, but in fact inhibited GVHDinduced by allogeneic CD4+ and CD8+ T cells(S20-S22). Other labs havedemonstrated that infusion of fully allogeneic DNT cells did not causeGVHD and could facilitate HSPC engraftment in mouse models of allogeneicbone marrow transplantation studies(S23)). In addition to suppressingunwanted GVHD, DNT cells have potent anti-cancer activity. The Zhang labdemonstrated that injection of mouse DNT cells rescued the recipientsfrom a lethal dose of lymphoma cells(S24). The mechanisms by which mouseDNT cells suppress allogeneic immune responses and kill cancer cellshave been studied extensively by the Zhang lab and others.

Safety Studies of Human Allogeneic DNT Cells

We and others have demonstrated that injection of allogeneic mouse DNTcells does not cause GVHD and can inhibit GVHD induced by infusion ofallogeneic CD4+ and CD8+ T cells (S20, S22, S23). Similar to mouse DNTs,human DNTs inhibited proliferation of CD4+ and CD8+ T cells in vitro(FIG. 8 a ). In patients who received allogeneic HSCT, a higherfrequency of DNTs was associated with a reduced severity of GVHD(525,S26), suggesting a beneficial effect of DNT cells. To validate the roleof ex vivo expanded human DNT cells in the context of GVHD, we used thestate-of-the-art xenograft models. All immune deficient NSG miceinjected with bulk human PBMC developed lethal xeno-GVHD(527-529), whilenone of the mice injected with DNT cells developed GVHD, measured bybody weight (FIG. 8 b ) and confirmed by histology of GVHD target organssuch as the liver, skin, intestine and lungs (FIG. 8 c ). Furthermore,treatment with DNTs significantly prolonged survival of NSG mice fromlethal xeno-GVHD caused by human PBMC (FIG. 8 d ). Collectively, thesedata suggest the potential of using DNTs to modulate GVHD associatedwith allo-HSCT.

Given that DNTs were cytotoxic to primary CD34+leukemic cells in vitro,and able to reduce leukemic engraftment in vivo8, it was critical toassess the potential cytotoxicity of DNTs against normal cells. An idealexperiment would be to test whether DNTs are cytotoxic to normal CD34+stem cells. Since there is no definitive cell surface marker for sortingnormal versus leukemic CD34+ cells, alternatively, healthy donor BMcells were co-cultured in vitro with ex vivo expanded DNTs. In bothCD33+myeloid and CD34+HSPC enriched populations, no differences in cellviability were detected between the DNT-treated and control groups after12 and 24 hours co-culture (FIG. 9 a ). Importantly, allogeneic DNTcells expanded from healthy donors are not cytotoxic to normal PBMC(FIG. 9 b ) or CD34+CD133+human hematopoietic stem/progenitor cells(HSPC) in vitro (FIG. 9 c ). To further assess the effect of DNTs onnormal BM cells in vivo, healthy donor BM cells were injected into NSGmice, followed by PBS or autologous DNT treatment. Five days after DNTinjection, the total numbers of viable human CD34+ cells were measured,and again no differences were observed between the control and treatmentgroups (FIG. 9 d ).

To further assess the potential effect of DNTs on normal hematopoieticcell differentiation and on mature hematopoietic cells in vivo, NSG micewere injected with normal HSPCs, and eight weeks later, mice weretreated with DNTs or PBS. The engraftment frequency and thedifferentiation of human hematopoietic cells into different lineageswere then compared between the two groups. Both the frequency (FIG. 9 e) and the lineage composition (FIG. 9 f ) of human hematopoietic cellswere comparable between DNT— and PBS— treated groups in PB, spleen andBM, indicating that DNTs do not alter the differentiation ofhematopoietic cells in vivo. Moreover, the frequency of HSPC enrichedCD34+ cells in BM (FIG. 90 was similar between the two treatment groups,further supporting the notion that ex vivo expanded allogenic DNTs donot target normal HSPCs. Taken together, these data indicate that DNTsare not cytotoxic to normal BM cells nor affect healthy donor stem cellengraftment or differentiation in xenograft models. These findings, forthe first time, point to the safety and potential efficacy of ex vivoexpanded DNTs as a novel immunotherapy to treat AML patients inchemotherapy-induced remission to decrease disease relapse and increasepatient survival.

Therapeutic Indications

In view of the above examples, applications in the human context areexpected to be validated, and include without limitation, the following.

To prevent or treat GVHD, e.g. when a patient is undergoing treatmentfor a disease where stem cell transplant is useful (e.g. for multipleMyeloma, . . . etc.), the patient could be provided DN Tregs subsequentto a stem cell or a cord blood cell transplant. This can be advantageousas it is after ablation of the patient's immune system. Alternatively orin combination, the patient could be provided DN Tregs prior to the stemcell or cord blood cell transplant. The DN Tregs administered could beallogenic or autologous.

To prevent or treat GVHD with leukemia, where stem cell and/or cordblood transplant forms a part of the treatment, the patient could beprovided DN Tregs after chemotherapy/radiation treatment (forantileukemic effect). Alternatively or in combination, the patient couldbe provided DN Tregs after induction chemotherapy/radiation treatmentand before stem cell or cord blood cell transplant (preferably afterablation of patient's own immune system). Alternatively or incombination, the patient could be provided DN Tregs after orconcurrently with stem cell /cord blood transplant. Again, the DN Tregsadministered could be allogenic or autologous.

Given the examples provided, it is expected that αβ-TCR⁺⁻ and γδ-TCR+DNTregs would be useful in these human indications.

Although preferred embodiments of the invention have been describedherein, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims. All documents disclosedherein, including those in the following reference list, areincorporated by reference.

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What is claimed is:
 1. A method for expanding human CD4⁻ CD8⁻ regulatoryT cells (DN Tregs) from a population of cells comprising DN Tregs,comprising: depleting the population of CD4+ and CD8+ cells; activatingthe population with anti-CD3 antibodies; then culturing the populationof cells with artificial antigen presenting cells (APCs) that express atleast one cell surface adhesion molecule for immunological synapse, theat least one cell surface adhesion molecule comprising at least one ofCD54 and CD58 wherein the DN Tregs are αβ-TCR+CD56-, γδ-TCR+, or both.2. The method of claim 1, wherein the artificial APCs display anti-CD3antibodies.
 3. The method of claim 2, wherein the artificial APCsfurther express a co-stimulatory molecule, preferably at least one ofCD80, CD86, CD83, 4-1BBL, OX40L, ICOSL, CD40L, and CD28 antibody.
 4. Themethod of claim 2, wherein the artificial APCs do not express inhibitorymolecules.
 5. The method of claim 4, wherein the inhibitory molecule isPDL1, PDL2, B7H3, or B7H4.
 6. The method of claim 2, wherein theartificial APCs further express at least one of M-CSF, IL-6, IL-8, TGF-βand MIP-1a.
 7. The method of claim 2, wherein the artificial APCsfurther express IL-7, IL-15 and/or IL-2.
 8. The method of claim 1,wherein the artificial APCs comprise K562-based artificial APCs.
 9. Themethod of claim 8, wherein the artificial APC is one of the following:i. K562-mbIL15-41BBL, expressing 4-1BBL and membrane-bound IL-15; ii.aAPC clone #9, expressing CD64, CD86, 4-1BBL, truncated CD19, andmembrane-bound IL-21; iii. KT64/86, expressing CD64 and CD86; iv.aAPC-A2 clone 33, expressing HLA class I, CD80, and CD83; v. 7F11ECCE,expressing CD64 and 4-1BBL; vi. K562cs, expressing CD32, CD80, CD83,CD86, and 4-1BBL; or vii. aAPC clone #4, expressing CD64, CD86, 4-1BBL,truncated CD19, and membrane-bound IL-15.
 10. The method of claim 1,wherein the culturing is additionally in the presence of IL-2.
 11. Themethod of claim 1, wherein the culturing is additionally in the presenceof IL-7 and/or IL15.
 12. The method of claim 1 wherein the activating iswith anti-CD3 antibodies cross-linked, or otherwise attached, to asurface.
 13. The method of claim 1, wherein the activating is withsoluble anti-CD3 antibodies.
 14. The method of claim 1, wherein theactivating is sequentially with anti-CD3 antibodies cross-linked, orotherwise attached, to a surface and soluble anti-CD3 antibodies. 15.The method of claim 1, wherein the population comprises peripheral bloodmononuclear cells (PBMCs) or cord blood mononuclear cells (CMBCs). 16.The method of claim 1, further comprising incubating the expanded DNTregs with at least one inhibitor of the PI3K/AKT/mTOR pathway.
 17. Themethod of claim 16, wherein the at least one inhibitor is a mTORinhibitor, dual PI3K/mTOR inhibitor, AKT inhibitor, or Pan-class I andisoform-specific PI3K inhibitors.
 18. The method of claim 16 wherein theinhibitor is a Rapalog.
 19. The method of claim 18, wherein theinhibitor is rapamycin, deforolimus, emsirolimus, everolimus,ridaforolimus, temsirolimus or a mTORC1/mTORC2 dual inhibitor.
 20. Themethod of claim 19, wherein the inhibitor is rapamycin.
 21. The methodof claim 16, wherein the inhibitor is Wortmannin, LY294002, PKI-179, orAkt inhibitor IV.